In previous work in our group (Havlin and Tycko, PNAS 2005), we carried out the first solid state NMR studies of protein folding. These studies focussed on the 35-residue protein domain HP35, which is known to contain three alpha-helical segments in its folded state and to be thermally stable up to approximately 70 C. HP35 was chosen because it can be readily synthesized by standard solid-phase peptide synthesis methods and because it has been the subject of numerous previous studies by experimental techniques and by computer modelling. In this work, we examined the dependence of solid state NMR signals from selectively isotopically-labeled HP35 on chemical denaturant (GdnHCl) concentration in frozen glycerol/water solutions (glass transition temperature of roughly -70 C). At low denaturant concentrations, 13C NMR signals characteristic of a helical protein were observed, as expected. At higher denaturant concentrations, two quite unexpected observations were made: (1) In the "fully unfolded" state (7 M GdnHCl), NMR signals from the three helical segments were markedly different, indicating a high level of disorder in the third helical segment, a mixture of residual helix content and disorder in the second helical segment, and an apparently low degree of disorder but no helix content in the first helical segment. This contradicts the simple assumption that the unfolded state is a uniform random coil; (2) Near the unfolding midpoint (4.5 M GdnHCl), the three helical segments appeared to have progressed to different stages along their respective unfolding paths, i.e., denaturation of HP35 cab not be described by a simple two-state model in which only the fully-folded and fully-unfolded states coexist. This work demonstrated that solid state NMR measurements can indeed provide new information about protein folding.[unreadable] [unreadable] Of particular interest was the observation that the first helical segment of HP35 was apparently highly ordered (but nonhelical) in the unfolded state. This raises the question of what the conformation of this segment actually is in the unfolded state. We have addressed this question in FY2007 by carrying out solid state NMR measurements that directly probe backbone phi and psi torsion angles for a particular site (Valine-50) in the first helical segment. These measurements employ three techniques developed previously in our group, with abbreviated names CT-DQFD, DQCSA, and 2DEXMAS. Without going into details of these techniques, suffice it to say that each technique provides independent constraints on backbone phi and psi torsion angles. Multiple independent constraints allow us to fit the data to simple models for the distribution of phi and psi populations (i.e., simple representations of the structural disorder). When these techniques are applied to HP35 in its folded state, the data are fit best by a single conformation, very close to the helical phi and psi angles determined by crystallography for folded HP35. In the unfolded state (7 M GdnHCl), the combined data can not be fit by a single conformation. Instead, the data are well described by significant populations near two phi,psi pairs, namely -75,155 degrees and -115,75 degrees. The first of these (with approximately 33% of the population) corresponds to the polyproline II conformation that has been suggested by other groups to be a dominant conformation in unfolded proteins. The second (with approximately 67% of the population) is in the "transition region" between alpha-helical and beta-strand conformations, and was not anticipated. Interestingly, ab initio calculations of 13C chemical shifts indicate that these two conformations have similar chemical shifts, accounting for the observation of relatively sharp lines in solid state NMR spectra, which had suggested a high degree of structural order.[unreadable] [unreadable] The work described in the above paragraph represents the first application of quantitative solid state NMR methods for determination of site-specific structural parameters in an unfolded state. A manuscript describing this work is in preparation.[unreadable] [unreadable] Experiments described above probe a "thermodynamically unfolded" state. We are currently attempting to study "kinetically unfolded" states, i.e., structural states that are trapped out of equilibrium by rapid change of solvent conditions, followed by rapid freeze-quenching. HP35 is a challenging case, because the main folding transition is believed to occur in approximately 10 microseconds, too fast to be freeze-quenched. Nonetheless, we have constructed an apparatus that permits freeze-quenching in approximately 100 microseconds (by spraying a protein solution, initially heated above the unfolding temperature, into cold isopentane, using a 50-micron-diameter spray nozzle and high pressure pumps to create a very fine, high-velocity jet of solution). With this apparatus, we are currently investigating whether folding of HP35 may occur in two stages, consisting of an initial formation of secondary structure on the 10 microsecond time scale, followed by a slower stage in which tertiary contacts form and sidechain packing is optimized. Such a slow stage might be invisible to optical techniques that have been used to characterize HP35 folding kinetics, but may be visible in solid state NMR. We also plan to carry out kinetic folding studies of slower-folding proteins, including the 67-residue alpha-spectrin SH3 domain, which we have recently succeeded in synthesizing directly by solid-phase methods and which is therefore amenable to selective isotopic labeling.